Cyclooxygenase (COX) is the enzyme that mediates biosynthesis of prostaglandins (PGs) and thromboxanes from arachidonic acid, and whose inhibition underlies the effectiveness of a variety of anti-inflammatory drugs (Sharma and Sharma, 1997, Indian J. Exp. Biol. 35: 1025-1031; Morteau, 2000, Arch. Immunol. Ther. Exp. 48: 473-480; Llorens, 2002, J. Mol. Graph Model. 20: 359-371; Smith et al., 2000, Annu. Rev. Biochem. 69: 145-182; FitzGerald and Patrono, 2001, N. Engl. J. Med. 345: 433-442; Vane and Botting, 1998, Inflamm. Res. 47: S78-S87). COX activity originates from two distinct and independently regulated isozymes, COX-1 and COX-2 (Dannhardt and Kiefer, 2001, Eur. J. Med. Chem. 36: 109-126; Otto and Smith, 1995, J. Lipid Mediat. Cell. Signal. 12: 139-156; Oberle et al., 1998, Circ. Res. 82: 1016-1020). Cyclooxygenase-2 is the dominant source of PGs which mediate pain and inflammation, while COX-1 catalyzes the formation of PGs that subserve housekeeping functions, such as the maintenance of gastrointestinal (GI) integrity. Traditional non-steroidal anti-inflammatory drugs (NSAIDs) inhibit both COX-1 and COX-2. As a consequence of COX-1 inhibition, however, traditional NSAIDs have been found to have adverse gastrointestinal effects, including both direct and indirect irritation of the gastrointestinal tract.
The coxibs, selective inhibitors of COX-2, were designed to inhibit the major enzymatic source of the PGs which mediate pain and inflammation, while sparing COX-1-derived PGs, which contribute dominantly to gastric cytoprotection (FitzGerald and Patrono, 2001, N. Engl. J. Med. 345: 433-42). Two coxibs, rofecoxib (Bombardier et al., 2000, N. Engl. J. Med. 343:1520-8, 2 p following 8) and lumiracoxib (Schnitzer et al., 2004, Lancet 364:665-74) have been shown in controlled trials to reduce the incidence of serious gastrointestinal (GI) adverse effects when compared with traditional NSAIDs. Rofecoxib, however, has been associated with an excess of heart attack and stroke in patients receiving this drug (25 mg/day) in the Adenomatous Polyp Prevention on VIOXX® (APPROVe) trial, and has recently been withdrawn from the market (FitzGerald, 2004, N. Engl. J. Med. 351:1709-11). A similar excess in cardiovascular events has recently been reported with celecoxib, again in a trial designed to prevent colonic adenomas (www(dot)nih(dot)gov/news/pr/dec2004/od-17(dot)htm). Furthermore, evidence has emerged to link a structurally distinct coxib, valdecoxib, to a cardiovascular hazard (Ott et al., 2003, J. Thorac. Cardiovasc. Surg. 125: 1481-92), suggesting strongly that this increased cardiovascular risk is a class effect. Indeed, valdecoxib has also been recently withdrawn from the market (www(dot)fda(dot)govlbbs/topics/news/2005/NEW01171(dot)html).
All of the coxibs depress substantially prostacyclin (PGI2), leaving platelet COX-1-derived thromboxane A2 (TxA2) unaffected (McAdam et al., 1999, Proc. Natl. Acad. Sci. USA 96: 272-7; Catella-Lawson et al., 1999, J. Pharmacol. Exp. Ther. 289; 735-41). PGI2 is a COX-2-derived molecule. PGI2, the dominant product of arachidonic acid in macrovascular endothelial cells, is formed by prostacyclin synthase (PGIS) action on prostaglandin endoperoxide intermediates, which are produced catalytically by COX-2 (Moncada et al., 1976, Nature 263: 663-5). PGI2 exhibits properties of potential relevance to atheroprotection. Specifically, it inhibits platelet aggregation, vascular smooth muscle contraction and proliferation (Cheng et al., 2002, Science 296: 539-541), leukocyte-endothelial cell interactions (Della Bella et al., 2001, Prostaglandins 65: 73-83) and cholesteryl ester hydrolase (Gryglewski et al., 1995, Ann. N.Y. Acad. Sci. 748: 194-206; discussion 206-7). It also activates reverse cholesterol transport (Morishita et al., 1990, J. Clin. Invest. 86: 1885-91). Indirect evidence suggests that PGI2 protects against oxidant-induced tissue injury. Deletion of the PGI2 receptor (IP) or suppression of PGI2 biosynthesis augments cardiac injury caused by ischemia/reperfusion (Xiao et al., 2001, Circulation 104: 2210-5) or the anthracycline, doxarubacin (Dowd et al., 2001, J. Clin. Invest. 108: 585-90). PGI2 also limits the cardiovascular effects of thromboxane A2 (TxA2), the major COX-1 product of platelets (Cheng et al., 2002, Science 296: 539-541). The cardiovascular effects of TxA2 include: platelet aggregation (Thomas et al., 1998, J. Clin. Invest. 102:1994-2001), elevation of blood pressure (Qi et al., 2002, J. Clin. Invest. 110: 61-9; Francois et al., 2004, Hypertension 43:364-9) and acceleration of atherogenesis (Kobayashi et al., 2004, J. Clin. Invest. 114:784-94; Cayatte et al., 2000, Arterioscler. Thromb. Vasc. Biol. 20: 1724-8; Huo et al., 2003, Nat. Med. 9: 61-7). This last effect may be particularly pertinent to the “latent period” before the emergence of cardiovascular risk in the APPROVe study. Indeed, urinary thromboxane (Tx) metabolites increase during lesion development (Pratico et al., 2000, Blood 96: 3823-6) and thromboxane receptor (TP) antagonism retards atherogenesis in mice (Cayatte et al., 2000, Arterioscler. Thromb. Vasc. Biol. 20: 1724-8). Deletion of the IP accelerates the initiation and early development of atherosclerosis in two mouse models (Kobayashi et al., 2004, J. Clin. Invest. 114: 784-94; Egan et al., 2004, Science 360: 1954-7). In this context, COX-2-derived PGI2 acts to limit the interactions of leucocytes and platelets with the vasculature and the attendant oxidant stress, thereby disrupting atherogenesis (Kobayashi et al., 2004, J. Clin. Invest. 114: 784-94; Egan et al., 2004, Science 360: 1954-7). Furthermore, in contrast to laminar shear stress, turbulent flow, which can be caused by atherosclerotic lesions, fails to increase endothelial expression of COX-2 (Topper et al., 1996, Proc. Natl. Acad. Sci. USA 93: 10417-22). Thus, defective expression of COX-2/PGIS-dependent PGI2 may predispose to focal atherogenesis in vivo.
This last effect may be particularly pertinent to the “latent period” before the emergence of cardiovascular risk in the APPROVe study. Although blood pressure was elevated by 3-4 mm Hg as early as one month in patients receiving rofecoxib in APPROVe, the increase in cardiovascular risk evolved slowly over time, becoming first evident after 18 months of treatment, in only 1-2% of patients. Thus, a small minority of the patients, apparently initially at low risk of cardiovascular disease, proceeded to increase that risk to a point that culminated in clinical events (FitzGerald, 2004, N. Engl. J. Med. 351:1709-11). Suppression of the COX-2-derived PGI2 pathway would afford a mechanism by which the hazard of drug-induced thrombosis would relate to the patient's underlying risk of cardiovascular disease (FitzGerald, 2003, Nat. Rev. Drug Discov. 2: 879-90). Indeed, this is consistent with evidence of a cardiovascular hazard in two placebo controlled trials of valdecoxib, another member of the class, in patients undergoing coronary artery bypass grafting (Furberg et al., 2005, Circulation 111:249 Epub 2005 Jan. 17), a setting of hemostatic activation (Anderson et al., 2003, Scand. Cardiovasc. J. 37: 356-62).
Therefore, evaluating initial cardiovascular risk in patients contemplating COX-2 selective inhibitor therapy, and on-going evaluation of cardiovascular risk in patients undergoing COX-2 selective inhibitor therapy would be of great value in the continued use of COX-2 selective inhibitor compounds. Assessing the plateau or decrease in cardiovascular risk by non-invasive means after cessation of COX-2 inhibitor therapy, or in patients on antioxidant therapy, would also be of great value.
In general, interest in assessing cardiovascular risk has been increasing in recent years. Early identification of cardiovascular risk is a high priority to medical doctors, because it would allow early treatment intervention in the hope of precluding a cardiovascular event. Historically and currently, cardiovascular risk assessment has relied substantially on measures of certain molecules in the blood. For instance, Wilson et al. (1998, Circulation 97:1837-1847) developed an algorithm using total cholesterol, LDL cholesterol and blood pressure to predict coronary heart disease (CHD). Sharrett et al. (2001, Circulation 104:1108-1113) identified the combination of LDL cholesterol, HDL cholesterol, triglycerides and lipoprotein (a) for predicting degree of risk of CHD. In recent years, research has demonstrated that inflammation plays a major role in atherothrombosis. Consequently, markers of inflammation, such as C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), have also recently been identified as predictors of cardiovascular risk (Ridker, 2001, Circulation 103:1813-1818; Cesari et al., 2003 Circulation 108:2317-2322). Disadvantageously, relying on blood tests requires drawing blood, an invasive procedure. Ideally, cardiovascular risk assessment would rely on non-invasive measures of biological compounds that correlate with cardiovascular risk. Furthermore, the identification of new, independent measures of cardiovascular risk may lead to increased accuracy in risk stratification.
Isoprostanes (iP) are PG isomers that are produced by free radical attack on arachidonic acid in situ in membrane phospholipids (Morrow et al., 1992, Proc. Natl. Acad. Sci. USA 89:10721-5). Isoprostanes are chemically stable end-products of lipid peroxidation that are released by phospholipases, circulate in plasma and are excreted in urine (Awad et al., 1993, J. Biol. Chem. 268:4161-4169). U.S. Pat. No. 5,891,622 to Morrow et al. teaches a method of assessing oxidative stress by measuring the isoprostane, 8-epi-PGF2α in a biological sample, including urine. It is noted in the background section of this patent that 8-epi-PGF2α may have diagnostic potential for atherosclerosis. U.S. Pat. No. 6,727,075 to FitzGerald et al. teaches a method of measuring isoprostane biomarkers in a mammal suspected of having Alzheimer's disease. The isoprostane biomarkers disclosed include 8,12-iso-iPF2α-VI.
There is an unmet need in the art for compositions and methods of assessing cardiovascular risk using noninvasive assays to assess cardiovascular risk. This need is particularly apparent for subjects contemplating or already undergoing COX-2 selective inhibitor therapy; subjects who have ceased such therapy; subjects who are undergoing antioxidant therapy; or subjects lacking clinical evidence of cardiovascular disease. The present invention meets this need.